Change in the Gut Microbiota of Lactating Sows and Their Piglets by Inclusion of Dietary Spray-Dried Plasma in Sow Diets

This study aimed to investigate the effects of dietary spray-dried plasma (SDP) on the gut microbiota of lactating sows and their piglets. A total of 12 sows were randomly assigned to one of two dietary treatment groups in a completely randomized design. The treatments were a sow diet based on corn and soybean meal (CON), and a CON diet with an added 1% SDP. The sows were fed the dietary treatments from d 30 before farrowing to weaning (d 28). The fecal samples of three sows from each treatment and two of their randomly selected piglets were collected to verify their fecal microbiota. There were no differences in the alpha diversity and distinct clustering of the microbial communities in the sows and their piglets when SDP was added to the sow diets from late gestation to weaning. The fecal microbiota of the lactating sows and their piglets showed a higher relative abundance of the phylum Bacteroidota and genus Lactobacillus and Ruminococcus and showed a lower relative abundance of the phylum Bacillota and genus Bacteroides, Escherichia/Shigella, and Clostridium in the sows fed the SDP diet than those fed the CON diet. Overall, these results show that the addition of SDP to the sow diet during lactation altered the gut environment with positive microbial composition changes. These results were similar in the nursing piglets, suggesting that the control of the sow diets during lactation may contribute to the intestinal health and growth in piglets after weaning.


Introduction
Sow feeding management during farrowing and lactation is an important nutritional strategy for pig production [1].The nutritional status during gestation affects the sow placental quality and fetal development [2][3][4].Previous studies have reported that the sow gestation diet supplemented with amino acids such as arginine and glutamine increased the placental blood supply and total litter weight at birth [3,5].Moreover, the farrowing and lactation period are physiologically important times because the hormones and metabolites related to milk production are modified by the nutritional changes in sow [6].Therefore, the nutritional management of sows during lactation is a major factor in determining the quality and yield of breast milk, which modifies the intestinal environment of suckling piglets [7].
The gastrointestinal tract (GIT) of neonatal piglets is underdeveloped at birth and the neonatal gut microbiota is maternally independent.The diarrhea of piglets after weaning is increased by GIT disorders and pathogenic microbial infections, which causes economic loss in the swine industry.The gut microbiota of neonatal piglets is established by the environment with their sow and the various bacteria and prebiotic compounds in the breast milk impact the intestinal development [1,8,9].In suckling piglets, Lactobacilli, which use oligosaccharides in the milk as an energy source, is established in the intestine and remains a predominant microbiota [9,10] .The growth of Lactobacilli protects the neonatal piglets from intestinal disease such as diarrhea by inhibiting the growth of pathogenic bacteria [11,12].Thus, the gut microbiota composition of neonatal piglets is correlated with the breast milk composition, which is determined by the nutrients in the sow lactation diets.
Dietary spray-dried plasma (SDP) is a by-product of livestock produced after slaughtering porcine, bovine, and This study aimed to investigate the effects of dietary spray-dried plasma (SDP) on the gut microbiota of lactating sows and their piglets.A total of 12 sows were randomly assigned to one of two dietary treatment groups in a completely randomized design.The treatments were a sow diet based on corn and soybean meal (CON), and a CON diet with an added 1% SDP.The sows were fed the dietary treatments from d 30 before farrowing to weaning (d 28).The fecal samples of three sows from each treatment and two of their randomly selected piglets were collected to verify their fecal microbiota.There were no differences in the alpha diversity and distinct clustering of the microbial communities in the sows and their piglets when SDP was added to the sow diets from late gestation to weaning.The fecal microbiota of the lactating sows and their piglets showed a higher relative abundance of the phylum Bacteroidota and genus Lactobacillus and Ruminococcus and showed a lower relative abundance of the phylum Bacillota and genus Bacteroides, Escherichia/Shigella, and Clostridium in the sows fed the SDP diet than those fed the CON diet.Overall, these results show that the addition of SDP to the sow diet during lactation altered the gut environment with positive microbial composition changes.These results were similar in the nursing piglets, suggesting that the control of the sow diets during lactation may contribute to the intestinal health and growth in piglets after weaning.
chicken, and is considered a feed additive because it contains growth factors such as fibrinogen, albumin, glycoprotein, and immunoglobulin [13].Moreover, SDP is currently being considered as an alternative to antibiotics in livestock feed because of its antimicrobial effect.Several studies have reported that weaning diets with the addition of SDP increased the number of Lactobacilli and reduced the Escherichia coli population in the ileal and cecal digesta and blocked the adhesion of pathogenic bacteria to the intestinal epithelium [14][15][16].Thus, we hypothesized that the feeding a diet with added SDP to sows during late farrowing and lactation would directly correlate with changes in the gut microbiota of the sows and the establishment of the gut microbiota in their piglets.Therefore, the objective of this study was to investigate the effects of lactation diets with an added 1% SDP on the gut microbiota of lactating sows and their litters.

Animals, Diets, and Study Design
A total of 12 lactating sows (Yorkshire × Landrace) with an average body weight (BW) of 227.78 ± 2.16 kg and average parity of 2.0 were used in this experiment.The sows were randomly allotted to one of two dietary treatments at d 84 of gestation based on a completely randomized design.On d 109 of gestation, the sows were moved from gestation crates to individual farrowing crates with free access to water.The dietary treatments were a typical diet based on corn and soybean meal as the control diet (CON) and the CON diet with an added 1% SDP.All dietary treatments were formulated to meet or exceed the national research council (NRC) requirements of gestating and lactating sows [17] (Table 1).Sows were fed 3.0 kg of the dietary treatments from d 84 of gestation until farrowing and lactation before weaning with free access to the diet and water.

Sample Collection and Preparation for Microbial Analysis
Three sow fecal samples per group were collected using rectal palpation at d 109 of gestation (before farrowing, -d 5), d 7, and d 28 (at weaning) of lactation.The feces of their litters were collected on d 7 and d 28 (at weaning) of lactation.The litter fecal samples of each sow were collected from two randomly selected piglets (one male and one female) by rectal palpation.The fecal samples were stored at -20°C until analysis.The DNA in each fecal sample was extracted using the Powerfood Microbial DNA Isolation kit (Mo Bio Laboratories, Inc., USA) according to the manufacturer's instructions.The concentration and quality of the isolated DNA were assessed using NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA) and stored at -20°C for analysis.

Fecal Microbiota Analysis by 16S rRNA Gene Sequencing
Each isolated genomic DNA sample was adjusted to a concentration of 1 ng/μl, and the targeting V3-V4 region of the 16s rRNA microbial genes were amplified by the polymerase chain reaction (PCR) step using specific primers as listed previously [18].Sequenced primers were designed based on 16S rRNA metagenomic sequencing library [19].By PCR, Bakt 341F (5-CCTACGGGNGGCWGCAG-3) and Bakt 805R (5-GGACTACHVGGGTWTCTAAT-3) with barcodes were amplified [19].The PCR product purification was performed by Wizard SV Gel and PCR Clean Up System (Promega, USA).The purified PCR product was confirmed by 1% agarose gel electrophoresis.The amplicon were sequenced by Illumina MiSeq platform in a commercial company (Macrogen Inc., Republic of Korea).Raw sequence data were processed by the Mothur software, and low-quality sequences were eliminated [20].Sequencing errors and chimeras were eliminated using UCHIME during Mothur processing [18].The remaining high quality sequences were categorized into operational taxonomic units (OTUs) clustering according to an identity cutoff of 97% [21].Bacterial taxonomic composition and community for each sample were performed using UCLUST [22] and Quantitative Insights into Microbial Ecology (QIIME) [23] based on ribosomal database project classifier for 16S rRNA.Alpha diversity was calculated using OTUs, Chao 1, Shannon, and Simpson indices.The principal coordinate analysis (PCoA) of beta diversity was performed by UniFrac distance metrics in QIIME to relate the bacterial community between dietary treatments.The taxonomic composition of each sample at phylum, family, and genus levels was shown as a percentage based on relative abundance.

Statistical Analysis
Alpha diversity data were expressed as mean ± standard deviation.Microbiota data were analyzed by Student's t-test using GraphPad Prism 9 (USA) with P < 0.05 considered as statistical significance.

Microbial Diversity of the Gut Microbiota of Lactating Sows and Their Piglets Using 16S rRNA Gene Sequencing
Fecal samples from the sows and their piglets were analyzed for alpha diversity using 16S rRNA gene sequencing.The six fecal samples from the sows in each dietary treatment group were collected on d 109 of gestation (before farrowing, -d 5), d 7, and d 28 (at weaning) of lactation (Table 2).The total of bacterial sequencing reads were 7,475.67 ± 3,317.83before farrowing (-d 5), 4,968.33 ± 1,758.36 at d 7 of lactation, and 3,601.00± 528.37 at weaning (d 28) with no difference between the dietary treatment groups.Moreover, the differences in the alpha diversity analysis such as the OTUs, Chao 1, Shannon, and Simpson index observed between the CON and SDP dietary treatment groups at each period were not statistically significant.The 12 fecal samples of the piglets in each sow group were collected on d 7 of lactation and d 28 at weaning (Table 3).The total bacterial sequencing reads were 4,389.50± 1,157.98 at d 7 of lactation and 4,290.67 ± 2,392.39 at weaning (d 28), but the piglets in the CON  group had higher (2613.67 ± 838.39 vs. 1775.83± 319.59, p < 0.05) average sequencing reads on d 7 of lactation than the piglets in the SDP group.However, there were no differences in OTUs, Chao1, Shannon, and Simpson index observed between the CON and SDP dietary treatment groups.Fecal microbial communities of the sows based on PCoA plots of the weighted UniFrac distances showed extensive clustering between the CON and SDP groups on d 7 of lactation (Fig. 1).However, there were no differences in the PCoA plot of the weighted UniFrac distances between the piglets from both dietary treatments (Fig. 2).

Discussion
Several studies have reported improvements in growth performance and intestinal health when SDP was used as a feed additive [14,16,24].In a recent study, the effects of spray-dried porcine plasma as a prebiotic were confirmed by changes in the intestinal microbiota [25].The addition of SDP increased the number of Lactobacilli, which was accompanied by the suppression of pathogenic bacteria and reduced the population of E. coli in the ileal and cecal digesta .We hypothesized that the addition of SDP to the diets of lactating sows would alter the gut microbial composition of the sows and their piglets.This is because the gastrointestinal tract of neonatal piglets is maternally independent, whereas the gut microbiota composition is influenced by the initial environment of the sow, such as the vagina, feces, and skin.
Our PCoA analysis of the fecal microbial communities of the sows showed a distinct separation between the CON and SDP groups during lactation.This indicates that the gut microbial composition of sows was affected by the dietary treatment with the addition of SDP.In our previous SDP study of sow productivity, we confirmed the results of improved productivity and immune response [13].This evidence supports the results that feeding a SDP additive diet to sows is related to sow productivity by positively altering the composition of the gut microbiota.This study demonstrated that a high relative abundance of the phylum Bacteroidota was present in the fecal samples from sows and their piglets in the SDP group.Bacteroidota are recognized as bacteria with the ability to digest otherwise indigestible dietary compounds to provide additional energy to the host [26].Moreover, several studies have reported that Bacteroidota produce short-chain fatty acids (SCFAs), which play an important role in gut health, provide energy to the intestinal mucosa, and promote the growth of epithelial cells [27,28].The relative abundance of the phylum Spirochaetes was also increased when the sows were fed the SDP diet during lactation.Spirochaetes have been reported to be related to the digestibility of dietary fiber because they break down hemicelluloses such as cellulose and xylan [29,30].The fecal microbial composition of the piglets in the SDP group showed a high relative abundance of the phylum Bacteroidota, similar to the results of the sows.These results suggest that the microbial composition of the sows can be transferred to their piglets and positively affect the digestibility of dietary fiber.
It was observed that the sow microbiota in the SDP group had a significantly higher relative abundance of the family Lactobacillaceae and genus Lactobacillus.They are bacteria well known for the production of lactic acid during carbohydrate fermentation [31][32][33].Previous studies have confirmed that Lactobacillus species have specific probiotic traits which act against pathogenic microorganisms such as E. coli in the intestine [34][35][36] .Our results also showed that sows fed the SDP diet during lactation had an increased relative abundance of the family Erysipelotrichaceae and genus Ruminococcaceae.The enteric Erysipelotrichaceae family has been reported to be associated with host lipid metabolism in nutritional studies [37,38].Generally, Ruminococcaceae are recognized as butyrate-and other SCFA-producing bacteria with functional capacities related to maintaining intestinal health [39,40].Our results suggest that the pathogenic microbial communities such as the genus Bacteroides, Escherichia/ Shigella, and Clostridium were decreased with the increase in these positive microbial communities.
Although a previous study reported the presence of bacteria in the placenta and amniotic fluid [41], it is unclear whether the early development of the gut microbiome of piglets is influenced by the intrauterine microbiome during pregnancy or postnatal environment.Based on previous studies, two hypotheses for the formation of the initial gut microbial colonization of piglets were confirmed.First, it was shown that the gut microbiota of the sow is deeply involved in the formation of the piglet's gut microbiota and consequently affects the immune system development, growth performance, and survival of the piglets [42][43][44].Another hypothesis is that the caring environment plays a crucial role in the function and composition of the gut microbiota during both the suckling and weaning periods [42,45].Our previous studies confirmed that the nutritional changes in sow diets affected the sow and piglet health and gut microbiota [46,47].The present study observed similar gut microbial profiles, with increases in the genus Lactobacillus and Ruminococcus and decreases in the genus Bacteroides, Escherichia/ Shigella, and Clostridium, in both the sows and piglets when the sows were fed a SDP additive diet during lactation.Therefore, our results suggest that the nutritional changes during lactation, such as SDP intake in sows, affect the development of the gut microbiome of piglets.

Conclusion
In conclusion, we observed that the gut microbiota composition of lactating sows was modified by the addition of 1% SDP to the sow diet from late gestation until weaning.In addition, it was confirmed that the changes in the composition of the gut microbiota of the piglets changed in a similar pattern to that of the sows.As such, this data suggests that the addition of SDP to the sow diet during lactation altered the gut environment with positive microbial composition changes.We believe that these results provide improved nutritional control of sow diets during lactation which may contribute to the intestinal health and growth of piglets after weaning.

Fig. 3 .
Fig. 3. Taxonomic classification of the total bacteria based on the 16S rRNA gene sequences at the phylum (A), family (B), and genus (C) levels for the sow fecal samples.n = 3; CON, control diet; SDP, CON added with 1% dietary SDP.

Fig. 4 .
Fig. 4. Taxonomic classification of the total bacteria based on the 16S rRNA gene sequences at the phylum (A), family (B), and genus (C) levels for the piglet fecal samples.n = 3; CON, control diet; SDP, CON added with 1% dietary SDP.